Proof of Concept
In vitro transcription and translation (TXTL) is a convenient cell-free system that has increasingly been developed to apply in synthetic biology1,2. In addition to achieve biosafety level, TXTL becomes powerful in prototype characterization of genetic parts, devices and circuits. Moreover, TXTL is particularly useful to express and purify proteins which are toxic, insoluble or unstable in cell-based system. Furthermore and amazingly, Dr. Vincent Noireau’s lab has demonstrated cell-free TXTL application in infectious bacteriophage production, in which T7 phage (40kbp, 77 genes, dsDNA) and T4 phage (170kbp, 289 genes, dsDNA) genome replication, synthesis, assembly can be performed in vitro just in a single test tube3,4.
In order to engineer Salmonella phage, we set up protocols including phage genome extraction (NOTEBOOK) and TXTL system (MEASUREMENT) based on the materials and procedures in Dr. Noireau’s paper. Because of some chemicals and biosafety level of Salmonella, we performed all of the related experiments at P2 laboratory of Prof. Ming-Shiou Jan at Chung Shan Medical University in Taiwan and conducted the experiments under supervision of him or his doctoral students.
We extracted the genomic DNAs of two of our isolated Salmonella phages and confirmed the gDNA integrity by running electrophoresis on 0.7% agarose gel (Fig. 1a). No plaque was observed using the extracted DNAs in the plaque assay (Fig. 1b), indicating no live phage contamination is in isolated gDNA extracts. Further, the extracted phage gDNA is able to generate infectious particles in Salmonella plaque assay after subjected to TXTL (Fig. 1c), demonstrating phage DNA can be expressed, assembled and packaged in vitro. Taken together, we can perform phage gDNA extraction and genome packaging in TXTL in our lab and ready to engineer reporter phages.
Figure 1 |Salmonella gDNA extraction and packaging in TXTL. (a) The integrity of DNA isolated from two Salmonella enterica serovar Typhimurium phages (named ST1 and ST2) was checked by electrophoresis on 0.7% agarose gel. (b,c) Extracted DNA as a control or DNA in TXTL reaction was spread on the lawn of Salmonella culture in plaque assay.
Tol2 transposon system is highly used in zebrafish transgenesis. The transposase protein (TPase) is from the Medaka fish (Oryzias latipes) aka Japanese rice fish, which catalyzes the transposition of the Tol2 elements through cut-and-paste mechanism. The minimal transposable Tol2 sequence (mTol2) contains 200-bp left arm and 150-bp right arm5. Up to 11kb DNA insert between Tol2 sequence can be integrated into the genome of nearly all vertebrates including zebrafish, frog, chicken, mouse, and human6.
A further application in synthetic biology was demonstrated by Jun Ni, et. al. 7, in which the recombinant TPase protein is fully functional in HeLa cell line and Zebrafish germline cells. In addition, the TPase can be expressed under T7 promoter in E. coli BL21 and purified with N-terminal 6xHis tag. The transposase is active in vitro and mediated the integration of DNA fragments between plasmids with Tol2 elements.
To engineer an isolated phage genome with unknown full genome sequence in our project, we want to create Tol2 transposon system. The system contains a HELPER (Tol2 tranposase, TPase), a DONOR (Tol2 mobile element) which becomes a vector to carry CARGO (phi29 DNA polymerase) jumping into a target (Salmonella phage genome). The engineered phage genome with recombinant phi29 DNA polymerase gene will be synthesized and packaged in TXTL reaction to acquire the infectious Salmonella phages. And we called it deSALMONEtor, which is a bio-detector that can specifically infect Salmonella spp. and report strong signals through RCA and DNA staining.
Oryzias latipes Tol2 transposase (TPase) gene sequence was taken from UniProt database8 and optimized based on E. coli codon preference. The TPase gene was designed with 6xHis tag and a GS linker at N-terminus and synthesized by Integrated DNA Technologies, Inc. (IDT). The DNA fragment was cloned into pSB1C3 as a basic BioBrick part (TPase/pSB1C3, Part:BBa_K3728000). The part was checked by colony PCR and restriction enzymes and further confirmed by sequencing (Fig. 2).
Figure 2 | 6xHis-GS-TPase/pSB1C3 construct check. DNAs were run electrophoresis on 1% agarose gel with 1kb marker. (a) 4 colonies were subject to PCR with TPase-specific forward and reverse primers (PCR product size: ~2000 bp). (b) The DNAs were extracted and digested by EcoRI and BamHI (2893, 870 and 235 bps).
His-tagged Tol2 transposase (TPase) was assembled with a T7 promoter and expressed in TXTL reaction with the bacterial cytoplasmic extracts prepared from IPTG-induced E. coli Rosetta 2(DE3) cells. The His-tagged proteins were further purified through Nickel column. The protein concentration was measured and analyzed on SDS-PAGE and Coomassie Blue Staining (Fig. 3). The protein was shown at around 70 kDa as the same size as the predicted TPase protein (664 a.a., 75 kDa). The Elution #4, #5 and #6 were collected and used for further studies.
Figure 3 |His-Tol2 transposase was expressed in TXTL and purified by Nickel column. 10 μg of protein lysates were analyzed by SDS-PAGE and Coomassie Blue Staining using 4–12% gradient gel (NuPAGE™, Thermo Fisher Scientific Inc.) Lane: (1) PageRuler™ Prestained Protein Ladder, (2) E. coli Rosetta 2(DE3) cell extracts (no DNA control), (3) total lysates in TXTL, (4) flow-through, (5) wash-through, (6) Elution #4, (7) Elution #5, (8) Elution #6, (9) Elution #7, (10) Elution #8, (11) Elution #9.
Minimal Tol2 transposable element (mTol2) has been characterized that is composed of 200-bp left arm and 150-bp right arm5. The 19-bp to 11-kbp DNA inserts between the arms can be excised and transposed efficiently by Tol2 transposase (TPase). Therefore, we’d like to make a BioBrick compatible vector based on Tol2 mobile element (pTol2), which can be further assembled through a BioBrick standard EcoRI-XbaI-SpeI-PstI rule.
We obtained the backbone of pBSII-SK-mTol2-MCS from Addgene (Plasmid #51817), which was given by Elly Tanaka9. We deleted restriction enzyme sites in MCS and generated novel BioBrick Prefix (EcoRI-NotI-XbaI) and BioBrick Suffix (SpeI-NotI-PstI) elements in the both ends by PCR. The resulting DNA plasmid backbone called pTol2 (Part:BBa_K3728002) was further assembled with the Part BBa_J04450 (i.e., the iGEM official standard insert on pSB1C3). The resulting J04450/pTol2 (Part:BBa_K3728003) was checked by PCR (Fig. 4a) and restriction enzymes (Fig. 4b) and also confirmed by sequencing.
Figure 4 | pTol2 and J04450/pTol2 constructs check. DNAs were run electrophoresis on 1% agarose gel with 1kb marker. (a) PCR producs of pTol2 (lane 1, 3429 bp) and BBa_J04450 (lane 2, 1112 bp). (b) 4 clones of J04450/pTol2 were checked by restriction enzymes (~ 3432 bp and ~1110 bp). Lanes 1-4 by EcoRI and SpeI. Lanes 5-8 by XbaI and PstI.
To test the TPase activity and Tol2 transposon system, we inserted a kanamycin resistance gene (KanR) cassette (Part:BBa_K3728004) and the reporters of ldhp-GFP-Tr(Part:BBa_K3728005), ldhp-RFP-Tr(Part:BBa_K3728006) and ldhp-amilCP-Tr(Part:BBa_K3728007) between the transposable elements on the pTol2 vector. ldhp is a constitutive and broad-host-range promoter, which was originally cloned and driving the lactate dehydrogenase gene in S. mutans. We have characterized the ldhp activities in S. mutans and E. coli in our project of iGEM 2020, as well as Salmonella and TXTL in this project of iGEM 2021. (for detail, go to check our (CONTRIBUTION page)
The GFP and RFP fluorescence intensities driven by ldhp on pTol2 vectors were measured at high level in TXTL reaction (Fig. 5). The strong GFP fluorescence can even be visualized by naked eyes under a Blue LED Illuminator. Compared the activities of ldhp to lac promoter (lacp), lacp is inhibited in TXTL because the extracts of E. coli Rosetta 2 (DE3) contains LacI repressor, which can be relieved by IPTG induction or using E. coli DH5α as extracts.
Figure 5 |Promoter activities on pTol2 vector in TXTL. GFP fluorescence was measured at Ex/Em = 500/530 nm using a microplate reader of BioTek Synergy H1. RFP was at Ex/Em = 586/611 nm. KanR/pTol2 in TXTX was set as a background control. AU means arbitrary unit. (a) ldhp-GFP-Tr/pTol2 activity in TXTL. The inset photo was captured under a blue LED light. (b) ldhp-RFP-Tr/pTol2 and J04450/pTol2 (i.e., lacp-RFP-Tr) in TXTL.
In vitro integration assay was used by Jun Ni, et al. to characterize the activity of purified recombinant Tol2 transposase (TPase) and the transposition of Tol2 mobile element7. We prepared the purified TPase from TXTL (Fig. 3) and performed PCR to generate KanR, ldhp-GFP-Tr and ldhp-amilCP-Tr (expressing blue color) DNA fragments flanked by 200-bp right and 150-bp left arms of pTol2. The mixtures of TPase, Tol2 mobile inserts and a target plasmid of pSB1C3 were incubated at 30°C for 2 hours. The resulting DNAs were cleaned up and subjected to transform E. coli DH5α competent cells. The colonies displaying kanamycin resistance, green fluorescence or blue color were counted as successful jumping to plasmids by active purified TPase. And the integration rate was calculated by comparing with chloramphenicol resistance or red colonies from pSB1C3 backbone carrying BBa_J04450 part (i.e., RFP coding device).
GFP/Tol2-integrated plasmid can transform E. coli to exhibit weak to strong green fluorescence in Fig. 6. Two plasmids of GFP-positive bacteria were extracted and checked by restriction enzymes. They are larger than pSB1C3 when single cut on the backbone by ApaLI (Fig. 7b). The schematic map of Fig. 7a showed the possible position of integration by a BamHI-cut on the insert and a ApaLI-cut on the backbone (Fig. 7c). The rate of successful integration was calculated by the ratio of numbers of KanR, GFP and BLUE colonies to CmR or RED colonies, respectively (Fig. 7d). The ratio was between 0.2% to 0.9%, of which data are consistent with the observation by Jun Ni, et al7. In sum, we can modify plasmid DNAs in vitro with an insert between Tol2 mobile elements (DONOR) and purified TPase enzymes (HELPER) from TXTL reaction.
Figure 6 |E. coli colonies on Cm agar plates were transformed by the mixture of GFP/Tol2 and pSB1C3 with TPase or without TPase as a control.
Figure 7 |Possible integration map and ratio. (a) Schematic maps showed the predicted integration sites. (b, c) pSB1C3::GFP/Tol2 Clone #1 (lane 1) and #2 (lane 2) or pSB1C3 as a control (lane 3) were cut by ApaLI on the backbone (b) or cut by ApaLI with a BamHI cut on the insert (c). DNA was analyzed by electrophoresis on 1% agarose gel with a 1kb marker. (d) The successful integration ratios are calculated by the numbers of colonies of pSB1C3::KanR/Tol2 on Kan agar plate divided by those of pSBC13 (CmR) on Cm agar plates or by the numbers of pSB1C3::GFP or pSB1C3::BLUE divided by colony numbers of pSB1C3 (RED) on Cm agar plates such as shown in Figure 6.
To engineer RCA reporter Salmonella phage, we requested Part:BBa_K3352001 (Φ29 DNA Polymerase with His-Tag and GS linker Sequence) from iGEM team TAS_Taipei. The part was assembled with ldhp promoter (BBa_K3376000) and a double terminator (BBa_B0015) onto the Tol2 mobile element vector (pTol2). The resulting composite part named ldhp-Phi29 DNA pol-Tr/pTol2 (BBa_K3728008) was checked by colony PCR, restriction enzymes and sequencing (Fig. 8).
Figure 8 |ldhp-Phi29 DNA pol-Tr/pTol2 construct check. DNAs were run electrophoresis on 1% agarose gel with 1kb marker. (a) 4 colonies were subject to PCR with ldhp forward primer and a reverse primer in the end of Phi29 gene (PCR product size: 1965 bp). (b) The DNAs were extracted and digested by EcoRI and PstI (3573 and 1973 bps).
Before we transform Salmonella, we need to check antibiotic sensitivity and find a suitable plasmid vector. We found our wild-type Salmonella strain obtained from the lab of Prof. Cheng-Yang Huang at Chung Shan Medical University is kanamycin and chloramphenicol sensitive but ampicillin resistant. Although pBR322-based vectors including BioBrick pSB6A1 and our pTol2 (BBa_K3728002) can replicate in Salmonella, all of the current vectors with pBR322 replication origin are carrying ampicillin resistance cassette (AmpR). Therefore, we were determined to make an improvement of the iGEM existing part of pSB6A1, in which AmpR was replaced by KanR (pSB6K1, BBa_K3728009)and CmR (pSB6C1, BBa_K3728010). Go to our IMPROVEMENT page or pSB6A1 part registry main page for the detail. However, unfortunately we can’t measure the phi29 DNA polymerase activity by transforming Salmonella cells with the plasmid of ldhp-Phi29 DNA pol-Tr/pTol2, which is ampicillin resistant.
To characterize the function of recombinant phi29 DNA polymerase, TXTL cell-free system can solve the problem caused by the difficulty in bacterial transformation or without suitable plasmid vectors. We took the DNA of ldhp- Phi29 DNA pol-Tr/pTol2 mixed into TXTL reaction with Salmonella extracts. The recombinant His-tagged phi29 DNA polymerase protein was purified through Nickel column and analyzed by SDS-PAGE and Coomassie Blue Staining (Fig. 9). The isolated proteins in Elution #4 and #5 were at the size of around 70 kDa as predicted (His-phi29 DNA polymerase: 590 amino acids, 68 kDa) and collected for the following studies.
Figure 9 |His-phi29 DNA polymerase was expressed in TXTL using Salmonella extracts and purified by Nickel column. 5 μg of protein lysates were analyzed by SDS-PAGE and Coomassie Blue Staining using 4–12% gradient gel (NuPAGE™, Thermo Fisher Scientific Inc.) Lane: (1) PageRuler™ Prestained Protein Ladder, (2) Salmonella cell extracts (no DNA control), (3) total lysates in TXTL, (4) flow-through, (5) wash-through, (6) Elution #3, (7) Elution #4, (8) Elution #5, (9) Elution #6, (10) Elution #7, (11) Elution #8.
Phi29 (Φ29) DNA polymerase is an exceptional processive polymerase possessing highly isothermal amplification and strong strand displacement activities10,11. The polymerase has been increasingly used in rolling circle amplification (RCA) assay that make long ssDNA strands using an initial primer and a single-stranded circular DNA template in the buffer containing dNTPs and Mg2+. The large amounts of amplified DNAs can be quickly generated within 1 hour and easily be measured with fluorescent DNA binding dye (e.g., EvaGreen® Dye).
We performed RCA by mixing a circular ssDNA and primer in the buffer with our purified phi29 DNA polymerase (Φ29) from TXTL using Salmonella extracts or a commercial recombinant Φ29 from New England Biolabs Inc. (NEB) as a positive control. The Φ29 enzymes were diluted with various factors in the assay. After incubation at 30°C for 1 hour, the RCA products were stained with EvaGreen DNA dye and subjected to a microplate reader to measure signals at Ex/Em=500/530 nm. And the fold changes in fluorescence intensity were calculated by dividing the values from Φ29-untreated groups (as controls). As shown in Fig. 10, a 12-fold change was achieved with our Φ29, indicating the functionality and the activity of ldhp-Phi29 DNA pol-Tr/pTol2 that are comparable to the commercial NEB Φ29 enzymes. Moreover, the green fluorescence can readily be seen with a blue led light, even using a 4 times diluted Φ29, proving the super high processivity of phi29 DNA polymerase in DNA amplification.
Figure 10 |RCA assay using TXTL-expressed (MINGDAO) or commercial (NEB) phi29 DNA polymerase (Φ29). The commercial Φ29 was purchased from NEB with a defined activity by units. The control was set without Φ29 treatment. The concentration and dilutions of enzymes were, respectively, 4, 2, 1 μg/μl for MINGDAO Φ29 and 1, 0.5, 0.25 units for NEB Φ29. The EvaGreen DNA binding signals were read at Ex/Em=500/530 nm in BioTek Synergy H1 Microplate Reader.
1. Marshall R, Noireaux V. Synthetic Biology with an All E. coli TXTL System: Quantitative Characterization of Regulatory Elements and Gene Circuits. Methods Mol Biol. 2018;1772:61-93. doi: 10.1007/978-1-4939-7795-6_4.
2. Tinafar A, Jaenes K, Pardee K. Synthetic Biology Goes Cell-Free. BMC Biol. 2019 Aug 8;17(1):64. doi: 10.1186/s12915-019-0685-x.
3. Shin J, Jardine P, Noireaux V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol. 2012 Sep 21;1(9):408-13. doi: 10.1021/sb300049p.
4. Rustad M, Eastlund A, Jardine P, Noireaux V. Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synth Biol (Oxf). 2018 Jan 22;3(1):ysy002. doi: 10.1093/synbio/ysy002.
5. Urasaki A, Morvan G, Kawakami K. Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition. Genetics. 2006 Oct;174(2):639-49. doi: 10.1534/genetics.106.060244.
6. Kawakami K. Tol2: a versatile gene transfer vector in vertebrates. Genome Biol. 2007;8 Suppl 1(Suppl 1):S7. doi: 10.1186/gb-2007-8-s1-s7.
7. Ni J, Wangensteen KJ, Nelsen D, Balciunas D, Skuster KJ, Urban MD, Ekker SC. Active recombinant Tol2 transposase for gene transfer and gene discovery applications. Mob DNA. 2016 Mar 31;7:6. doi: 10.1186/s13100-016-0062-z
8. Tol2 transposase sequence (Oryzias latipes) at UniProt: UniProtKB - Q9PVN3 (Q9PVN3_ORYLA)
9. Khattak S, Murawala P, Andreas H, Kappert V, Schuez M, Sandoval-Guzmán T, Crawford K, Tanaka EM. Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc. 2014 Mar;9(3):529-40. doi: 10.1038/nprot.2014.040.
10. Johne R, Müller H, Rector A, van Ranst M, Stevens H. Rolling-circle amplification of viral DNA genomes using phi29 polymerase. Trends Microbiol. 2009 May;17(5):205-11. doi: 10.1016/j.tim.2009.02.004.
11. Yue S, Li Y, Qiao Z, Song W, Bi S. Rolling Circle Replication for Biosensing, Bioimaging, and Biomedicine. Trends Biotechnol. 2021 Mar 11:S0167-7799(21)00039-1. doi: 10.1016/j.tibtech.2021.02.007.